Decades of research into the cellular and molecular mechanisms of hematopoietic stem cell (HSC) maintenance, proliferation, and differentiation have led to volumes of information about the hematopoietic stem cell. Despite this extensive knowledge, the identity of cells and cell products that provide the extrinsic control of HSC fate remain largely unknown, but are attributed to cells and cell products of the bone marrow microenvironment (ME). A Novel 3D culture system incorporating cells suspended in a matrix together with endothelial lined vessels provides a new way to interrogate cell interactions within the complex ME. Utilizing well characterized stromal cell lines that are functionally distinct, we will build a 3D ME to mimic the signaling thought to occur via cell to cell interactions in vivo. W will then introduce gene modified CD34+ cells transduced with a mCherry gene under the control of the megakaryocyte lineage specific promoter PF-4 to detect lineage commitment in these cells. Once the commitment assay is standardized, we will test the hypothesis that monocytes in contact with stromal cells play a critical role in defining the functionality of the M, particularly as it relates to sustaining the uncommitted state. We anticipate that monocytes together with specific stromal cells will provide signals that function to prevent commitment. To test the function of the non GFP+ and presumably uncommitted cells, we will remove the cells from the cultures and test their ability to produce multi-lineage progeny in expansion cultures and NOD/SCID assays. If we can demonstrate the maintenance of multi-potentiality, with or without cell division, future studies can use knockdown techniques to identify the pathways involved.